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WO2018183485A1 - Procédés d'isolement de séquences de récepteur de lymphocytes t spécifiques à un néo-antigène - Google Patents

Procédés d'isolement de séquences de récepteur de lymphocytes t spécifiques à un néo-antigène Download PDF

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Publication number
WO2018183485A1
WO2018183485A1 PCT/US2018/024828 US2018024828W WO2018183485A1 WO 2018183485 A1 WO2018183485 A1 WO 2018183485A1 US 2018024828 W US2018024828 W US 2018024828W WO 2018183485 A1 WO2018183485 A1 WO 2018183485A1
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WIPO (PCT)
Prior art keywords
tcr
cdna
sequences
multiple fragments
sequence
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PCT/US2018/024828
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English (en)
Inventor
Yong-Chen LU
Peter Fitzgerald
Zhili ZHENG
Steven A. Rosenberg
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority to CN201880022673.3A priority Critical patent/CN110612446B/zh
Priority to EP18721882.1A priority patent/EP3602053B1/fr
Priority to JP2019553512A priority patent/JP7317712B2/ja
Priority to US16/495,508 priority patent/US11898207B2/en
Priority to CA3057375A priority patent/CA3057375A1/fr
Priority to AU2018244371A priority patent/AU2018244371B2/en
Priority to ES18721882T priority patent/ES2957959T3/es
Priority to EP23176735.1A priority patent/EP4234576A3/fr
Priority to CN202310701331.7A priority patent/CN116726047A/zh
Application filed by The United States Of America, As Represented By The Secretary, Department Of Health And Human Services filed Critical The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Publication of WO2018183485A1 publication Critical patent/WO2018183485A1/fr
Priority to JP2023117842A priority patent/JP7569416B2/ja
Priority to US18/397,448 priority patent/US20240124935A1/en
Priority to JP2024174698A priority patent/JP2025020127A/ja

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    • A—HUMAN NECESSITIES
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4201—Neoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/30—Coculture with; Conditioned medium produced by tumour cells

Definitions

  • Adoptive cell therapy using cells that have been genetically engineered to express a cancer antigen (e.g., neoantigen)-specific T cell receptor (TCR) can produce positive clinical responses in some cancer patients.
  • a cancer antigen e.g., neoantigen
  • TCR tumor necrosis factor receptor
  • TCRs that specifically recognize cancer antigens e.g., neoantigens
  • TCRs may be difficult to identify and/or isolate from a patient. Accordingly, there is a need for improved methods of obtaining cancer-reactive (e.g., neoantigen-reactive) TCRs.
  • An embodiment of the invention provides a method of isolating paired T cell receptor (TCR) alpha and beta chain sequences, or an antigen-binding portion thereof, the method comprising: (a) isolating, from a biological sample, T cells having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation; (b) co- culturing the isolated T cells with antigen presenting cells (APCs) that present the mutated amino acid sequence so that the T cells express one or more T cell activation markers; (c) sorting the co-cultured T cells into separate single T cell samples; (d) isolating mRNA from each separate single T cell sample; (e) sequencing the mRNA from each separate single T cell sample, wherein the sequencing comprises: (i) producing cDNA from the mRNA and amplifying the cDNA; (ii) producing multiple fragments of the amplified cDNA and tagging the multiple fraginents; (iii) amplifying the
  • Another embodiment of the invention provides a method of automatically identifying the T cell receptor (TCR) alpha and beta chain V segment sequences and CDR3 sequences of a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: (a) receiving, at a user computing device, sequences of multiple fragments of cDNA, wherein the cDNA is encoded by mRNA produced by a single T cell following co-culture of the T cell with antigen presenting cells (APCs) that present the mutated amino acid sequence so that the T cell expresses one or more T cell activation markers; (b) perfonriing computerized alignment of the sequences of each of the multiple fragments of cDNA to a reference TCR sequence database to identify TCR alpha chain variable (V) segment sequences and TCR beta chain V segment sequences of the multiple fragments of cDNA; (c) perfonriing computerized identification of TCR
  • TCR T cell receptor
  • CDR3 complementarity detenriining region 3
  • Another embodiment of the invention provides a method of preparing a population of cells that express paired TCR alpha and beta chain sequences, or an antigen- binding portion thereof, the method comprising: isolating paired TCR alpha and beta chain sequences, or an antigen-binding portion thereof, according to any of the inventive methods described herein, and introducing a nucleotide sequence encoding the isolated paired TCR alpha and beta chain sequences, or the antigen-binding portion thereof, into host cells to obtain cells that express the paired TCR alpha and beta chain sequences, or the antigen- binding portion thereof.
  • a further embodiment of the invention provides a pair of TCR alpha and beta chain sequences, or an antigen-binding portion thereof, isolated according to any of the inventive methods described herein.
  • Still another embodiment of the invention provides an isolated population of cells prepared according to any of the inventive methods described herein.
  • compositions and methods of treating or preventing cancer are provided.
  • Figure 1 is a schematic illustrating a method of identifying neoantigen-reactive TILs.
  • Figure 2 is a schematic illustrating a method of identifying neoantigen-specific TCRs.
  • Figure 3 A and Figure 3B are graphs showing the percentage of IFN- ⁇ (A) and IL- 2 (B) reads within the total Rl reads measured in 4090 F7 T cells that were co-cultured with TMG-5-pulsed autologous DCs for 4 hr and then subjected to single-cell RNA-seq analysis.
  • Figure 3C is a graph showing the amount of IFN- ⁇ (pg/mL) secreted by donor T cells which were untransduced (unshaded bars) or transduced with the 4090 TCR (shaded bars) upon co-culture with DCs pulsed with TMG-5 or TMG-6. DCs pulsed with no TMG (“w/o") served as a negative control.
  • Figure 3D is a graph showing the amount of IFN- ⁇ (pg/mL) secreted by 4090 TCR-transduced T cells upon co-culture with 4090 DCs which had been pulsed with a mutated 25-mer peptide corresponding to one of the indicated minigenes from TMG-5.
  • Figure 3E is a graph showing the amount of IFN- ⁇ (pg/mL) secreted by 4090 TCR-transduced T cells upon co-culture with 4090 DCs which had been pulsed with the indicated concentration (cone.) ( ⁇ ) of purified 25-mer WT (open circles) or mutated (closed circles) USP8 peptide.
  • Figure 4 A and Figure 4B are graphs showing the percentage of IFN- ⁇ (A) and IL- 2 (B) reads within the total Rl reads measured in 4095 F5 T cells that were co-cultured with TMG- 1 -pulsed autologous DCs for 4 hr and then subjected to single-cell RNA-seq analysis.
  • Figure 4C is a graph showing the percentage of IFN- ⁇ and IL-2 reads within the total Rl reads measured in the single cell which expressed detectable IL-2 reads in Fig. 4B.
  • Figure 4D is a graph showing the amount of IFN- ⁇ (pg/mL) secreted by donor T cells which were untransduced (unshaded bars) or transduced with the 4095 TCR (shaded bars) upon co-culture with DCs pulsed with full-length WT or mutated KRAS mRNA. DCs pulsed with no peptide ("w/o") served as a negative control.
  • Figure 4E is a graph showing the amount of IFN- ⁇ (pg/mL) secreted by 4095 TCR-transduced T cells upon co-culture with 4095 DCs which had been pulsed with the indicated concentrations ( ⁇ ) of a purified 9-mer WT (open circles) or mutated (closed circles) KRAS peptide .
  • Figure 5A and Figure 5B are graphs showing the percentage of IFN- ⁇ (A) and IL- 2 (B) reads within the total Rl reads measured in 41 12 F5 T cells that were co-cultured with TMG-9-pulsed autologous DCs for 4 hr and then subjected to single-cell RNA-seq analysis.
  • Figure 5C is a graph showing the percentage of IFN- ⁇ and IL-2 reads within the total Rl reads measured in the 8 single-cells which expressed detectable IL-2 reads in Fig.
  • Figure 5D is a graph showing the amount of IFN- ⁇ (pg/mL) secreted by donor T cells which were untransduced (unshaded bars) or transduced with the 41 12 TCR (shaded bars) upon co-culture with DCs pulsed with TMG-9 or TMG-10. DCs pulsed with no peptide ("w/o") served as a negative control.
  • Figure 5E is a graph showing the amount of IFN- ⁇ (pg/mL) secreted by 41 12 TCR-transduced T cells upon co-culture with EBV-transformed B cells pulsed with one of the indicated pools (SPP-1 to SPP-10) of short peptides. EBV-transformed B cells pulsed with no peptide ("w/o") served as a negative control.
  • Figure 5F is a graph showing the amount of IFN- ⁇ (pg/mL) secreted by 41 12 TCR-transduced T cells upon co-culture with EBV-transformed B cells pulsed with SPP-9 or one of the indicated short peptides VWDALFADGLSLCL (SEQ ID NO: 18;
  • WRRVAWSYDSTLL (SEQ ID NO: 19 WSYDSTLL (SEQ ID NO: 20; WSYDSTLLA (SEQ ID NO: 21 ; WSYDSTLLAY (SEQ ID NO: 22; YLALVDKNIIGY (SEQ ID NO: 23; or YSEPDVSGK (SEQ ID NO: 24.
  • Figure 5G is a graph showing the amount of IFN- ⁇ (pg/mL) secreted by 41 12 TCR-transduced T cells upon co-culture with EBV-transformed B cells pulsed with purified mutated (closed circles) NBAS peptide WSYDSTLLAY (OS) (SEQ ID NO: 4) or its WT (open circles) counterpart.
  • Figure 6 is a block diagram illustrating a system in accordance with some embodiments of the invention.
  • Figure 7 is a block diagram illustrating components of a computing device according to an embodiment of the invention.
  • FIG. 8 is a flow diagram of method steps for automatically identifying the T cell receptor (TCR) alpha and beta chain V segment sequences and CDR3 sequences of a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, according to an embodiment of the invention.
  • TCR T cell receptor
  • Figure 9A is a graph showing the number of IFN- ⁇ positive spots detected after screening TIL4171F6 T cells against a library of 25-mer long-peptide pools (PP) encoding mutations in an ELISPOT assay.
  • T cells treated with OKT3 antibody served as a positive control.
  • T cells cultured with no peptide pool (w/o) served as a negative control.
  • Figures 9B and 9C are graphs showing the expression of IFN- ⁇ (FP M (Fragments Per Kilobase of transcript per Million mapped reads)) (Fig. 9B) and IL-2 (Fig. 9C) by TIL 4171 F6 T cells upon co-culture with PP-3-pulsed autologous DCs.
  • IFN- ⁇ FP M (Fragments Per Kilobase of transcript per Million mapped reads)
  • Fig. 9C IL-2
  • Figure 9D is a 2-dimensional scatter plot combining the data of Figures 9B and 9C showing the relationship of IFN- ⁇ and IL-2 expression in each single cell (each dot represents a single cell).
  • Figure 9E is a graph showing the amount of IFN- ⁇ (pg/mL) produced following co-culture of untransduced (unshaded bars) or 4171TCR-transduced (shaded bars) cells with PP-pulsed DC. T cells cultured with no peptide pool (w/o) served as a negative control.
  • Figure 9F is a graph showing the amount of IFN- ⁇ (pg/mL) produced following co-culture of 4171TCR- transduced T cells with DCs pulsed with the indicated peptides. T cells cultured with no peptide pool (w/o) served as a negative control.
  • Figure 9G is a graph showing the amount of IFN- ⁇ (pg/mL) produced following co-culture of 4171TCR-transduced T cells with DCs pulsed with the indicated concentration ( ⁇ ) of WT (open circles) or mutated (closed circles) SIN3A peptide.
  • An embodiment of the invention provides a method of isolating paired TCR alpha and beta chain sequences, or an antigen-binding portion thereof.
  • inventive methods may address any of a variety of different challenges to the identification and isolation of functional TCRs having the desired antigenic specificity.
  • TCRa and ⁇ chains may express two functional TCRa chains, while only one of the TCRa chains likely has the desired specificity.
  • inventive methods may provide any of a variety of advantages.
  • the inventive methods may significantly reduce the time and/or cost that is necessary to isolate and identify the sequence of a TCR that has antigenic specificity for a cancer antigen (e.g., a neoantigen) after a biological sample (e.g., tumor sample) is removed from a patient.
  • a cancer antigen e.g., a neoantigen
  • a biological sample e.g., tumor sample
  • host cells e.g., autologous T cells
  • the numbers of transduced cells may be expanded, and the expanded numbers of transduced cells may be administered to the patient for the treatment and/or prevention of cancer.
  • the inventive methods may (i) identify both the cancer antigen and the sequence of the TCR which recognizes the cancer antigen and/or (ii) facilitate highly personalized TCR therapy targeting cancer antigens (e.g., neoantigens).
  • cancer antigens e.g., neoantigens
  • the inventive methods may be, advantageously, less time-consuming, less laborious, and have a higher success rate as compared to methods of isolating paired TCRa/ ⁇ sequences using T cell cloning by limiting dilution.
  • the inventive methods may also make it possible to efficiently identify the correct pair of TCR alpha and beta chains in those T cells that have more than one functional TCRa gene.
  • the inventive methods may also identify and isolate paired TCR alpha and beta chain sequences (having the desired antigen specificity) from a highly diverse population of T cells.
  • the ⁇ TCR is a heterodimer composed of a and ⁇ protein chains. Each chain includes two extracellular domains, the variable (V) region and the constant (C) region, followed by a transmembrane region and a short cytoplasmic tail.
  • the variable domain of each of the TCR a-chain and ⁇ -chain have three "complementarity determining regions" (CDR1 , CDR2 and CDR3) which contact and recognize a peptide-MHC complexes.
  • CDR1 , CDR2 and CDR3 three "complementarity determining regions"
  • the a and ⁇ CDR3s are responsible for recognizing processed antigen. From T cell to T cell, there is an extremely high degree of polymorphism in the amino acid sequences of the CDR3a and CDR3 ⁇ .
  • polymorphism is necessary for T cells to recognize the wide scope of antigens that confront the immune system.
  • the polymorphism in the amino acid sequences of the CDR3a and CDR3 ⁇ result from DNA rearrangements within the TCR a and ⁇ genes that occur during the maturation of a T cell.
  • the genes that encode the TCR are made up of cassettes of coding sequence referred to as a "V segment” and a "J segment” in the TCR a-gene and a "V segment", a "D segment,” and a "J segment” in the TCR ⁇ -chain. Stochastic rearrangement in the genomic DNA results in the juxtaposition of these DNA segments resulting in a functional TCR gene. These rearrangements may be imprecise and junctions of the Va-Ja and ⁇ - ⁇ - ⁇ segments may be highly variable.
  • the CDR3 of the alpha chain is encoded by a portion of the V segment and all of the J segment.
  • the CDR3 of the beta chain is encoded by a portion of the V segment, all of the J segment, and all of the D segment.
  • the method may comprise isolating, from a biological sample, T cells having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation.
  • a biological sample is a tumor sample or a sample of peripheral blood.
  • biological samples include, without limitation, tissue from a primary tumors, tissue from the site of metastatic tumors, exudates, effusions, ascites, fractionated peripheral blood cells, bone marrow, peripheral blood buffy coat, and cerebrospinal fluid.
  • the biological sample may be obtained by any suitable means, including, without limitation, aspiration, biopsy, resection, venous puncture, arterial puncture, lumbar spinal puncture, shunts, catheterization, or the placement of a drain.
  • the T cells which are isolated from the biological sample have antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation.
  • antigenic specificity means that a TCR, or the antigen-binding portion thereof, can specifically bind to and immunologically recognize the mutated amino acid sequence encoded by the cancer-specific mutation.
  • the cancer-specific mutation may be any mutation in any gene which encodes a mutated amino acid sequence (also referred to as a "non-silent mutation") and which is expressed in a cancer cell but not in a normal, noncancerous cell.
  • the isolating of T cells having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation may comprise: identifying one or more genes in the nucleic acid of a cancer cell of a patient, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence; inducing autologous antigen presenting cells (APCs) of the patient to present the mutated amino acid sequence; co-culturing autologous T cells of the patient with the autologous APCs that present the mutated amino acid sequence; and selecting the autologous T cells that (a) were co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a major his
  • APCs autologous antigen presenting cells
  • the inventive method further comprises co-culturing those isolated T cells with APCs that present the mutated amino acid sequence so that the T cells express one or more T cell activation markers.
  • the APCs may include any cells which present peptide fragments of proteins in association with major histocompatibility complex (MHC) molecules on their cell surface.
  • MHC major histocompatibility complex
  • the APCs may include, for example, any one or more of macrophages, dendritic cells (DCs), langerhans cells, B- lymphocytes, and T-cells.
  • the APCs are DCs.
  • T cell activation markers may be used to identify those T cells having antigenic specificity for the mutated amino acid sequence.
  • T cell activation markers include, but are not limited to, any one or more of programmed cell death 1 (PD-1), lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3), 4-1 BB, OX40, CD107a, granzyme B, interferon (IFN)-y, interleukin (IL)-2, tumor necrosis factor alpha (TNF-a), granulocyte/monocyte colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.
  • PD-1 programmed cell death 1
  • LAG-3 lymphocyte-activation gene 3
  • TIM-3 T cell immunoglobulin and mucin domain 3
  • 4-1 BB OX40
  • CD107a granzyme B
  • IFN interferon
  • IL interleukin
  • the method further comprises sorting the co-cultured T cells into separate single T cell samples and isolating mRNA from each separate single T cell sample.
  • the sorting into separate single T cell samples and the isolating of mRNA may be automated.
  • the sorting into separate single T cell samples and the isolating of the mRNA may be carried out using a FLUIDIGM CI automated single-cell isolation and preparation system (available from Fluidigm, South San Francisco, CA).
  • the inventive method may, advantageously, provide any number of separate, single-cell mRNA samples (for example, about 2, about 3, about 4, about 5, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the foregoing values).
  • the method comprises preparing about 96 separate, single-cell mRNA samples.
  • the method may further comprise labeling the mRNA from each separate single T cell sample with a different tag (e.g., barcode) for each separate single T cell sample.
  • a different tag e.g., barcode
  • the mRNA from each separate single T cell sample may be labeled using the ILLUMINA NEXTERA XT DNA library preparation kit (available from Illumina, San Diego, CA).
  • the inventive method further comprises sequencing the mRNA from each separate single T cell sample.
  • the sequencing may be earned out in any suitable manner known in the art.
  • Preferred examples of sequencing techniques that may be useful in the inventive methods include Next Generation Sequencing (NGS) (also referred to as NGS) (also referred to as NGS)
  • NGS refers to non-Sanger-based high-throughput DNA sequencing
  • NGS nucleic acid templates may be randomly read in parallel along the entire genome by breaking the entire genome into small pieces.
  • NGS may, advantageously, provide nucleic acid sequence information from each separate single T cell mRNA sample in very short time periods, e.g., within about 1 to about 2 weeks, preferably within about 1 to about 7 days, or most preferably, within less than about 24 hours.
  • Multiple NGS platforms which are commercially available or which are described in the literature can be used in the context of the inventive methods, e.g., those described in Zhang et al., J. Genet. Genomics, 38(3): 95-109 (201 1) and Voelkerding et al., Clinical Chemistry, 55: 641-658 (2009).
  • Non-limiting examples of NGS technologies and platforms include sequencing- by-synthesis (also known as "pyrosequencing") (as implemented, e.g., using the GS-FLX 454 Genome Sequencer, 454 Life Sciences (Branford, CT), ILLUMINA SOLEXA Genome Analyzer (Illumina Inc., San Diego, CA), the ILLUMINA HISEQ 2000 Genome Analyzer (Illumina), or the ILLUMINA MISEQ system (Illumina) or as described in, e.g., Ronaghi et al., Science, 281 (5375): 363-365 (1998)), sequencing-by-ligation (as implemented, e.g., using the SOLID platform (Life Technologies Corporation, Carlsbad, CA) or the POLONATOR G.007 platform (Dover Systems, Salem, NH)), single-molecule sequencing (as implemented, e.g., using the PACBIO RS system (Pacific Biosciences (Menlo Park, CA
  • HELISCOPE platform Helicos Biosciences (Cambridge, MA)
  • nano-technology for single- molecule sequencing as implemented, e.g., using the GRIDON platform of Oxford
  • Nanopore Technologies (Oxford, UK), the hybridization-assisted nano-pore sequencing (HANS) platforms developed by Nabsys (Providence, RI), and the ligase-based DNA sequencing platform with DNA nanoball (DNB) technology referred to as probe-anchor ligation (cPAL)), electron microscopy-based technology for single-molecule sequencing, and ion semiconductor sequencing.
  • HANS hybridization-assisted nano-pore sequencing
  • DNS ligase-based DNA sequencing platform with DNA nanoball
  • cPAL probe-anchor ligation
  • the sequencing of the mRNA from each separate single T cell sample may comprise producing cDNA from the mRNA and amplifying the cDNA, producing multiple fragments of the amplified cDNA and tagging the multiple fragments, amplifying the tagged, multiple fragments of the cDNA, and sequencing the amplified, tagged multiple fragments of the cDNA.
  • the tagging may comprise adding a nucleotide sequence to each multiple fragment so that the multiple fragments can be distinguished from one another.
  • the sequencing identifies the sequences of each of the multiple fragments of cDNA.
  • the sequence of each of the multiple fragments of cDNA is also referred to as a "read.”
  • the sequencing of the mRNA may generate any number of reads.
  • the sequencing of the mRNA may generate about 1,000,000 reads, about 900,000 reads, about 800,000 reads, about 700,000 reads, about 600,000 reads, about 500,000 reads, about 400,000 reads, about 300,000 reads, about 200,000 reads, about 100,000 reads, or more, or a range defined by any two of the foregoing values, for each single T cell sample.
  • Rl and R2 may complement each other.
  • the method comprises measuring only Rl reads, only R2 reads, or both Rl and R2 reads.
  • Rl may have a higher sequencing quality than R2.
  • the method comprises measuring only Rl reads.
  • the method further comprises aligning the sequences of each of the multiple fragments of cDNA to a known sequence of the one or more T cell activation markers to identify which single T cell sample contained a single T cell which expressed the one or more T cell activation markers.
  • the one or more single T cell(s) which expressed the one or more T cell activation markers following co-culture with the APCs that present the mutated amino acid sequence encoded by a cancer-specific mutation are identified as expressing a TCR which has antigenic specificity for the mutated amino acid sequence encoded by a cancer- specific mutation.
  • the method further comprises aligning the sequences of each of the multiple fragments of cDNA to a reference TCR sequence database to identify TCR alpha chain variable (V) region sequences and TCR beta chain V region sequences of the multiple fragments of cDNA of each separate single T cell sample which was identified to express one or more T cell activation markers.
  • the sequences of each of the multiple fragments of cDNA are aligned against known TCR variable segment sequences in order to identify which cDNA fragments contain all or a portion of the variable segment sequence and to locate the approximate position of the 3' end of the variable segment sequence on the cDNA fragment(s).
  • the 3' end of the variable segment sequence indicates the approximate location of the CDR3.
  • the reference TCR sequence database may be any suitable reference TCR sequence database.
  • An example of a reference TCR sequence database may include sequences obtained from the international IMMUNOGENETICS information system (IMGT) database (//www.imgt.org), described in Lefranc et al., Nucleic Acids Res., 43: D413-422 (2015).
  • IMGT international IMMUNOGENETICS information system
  • the aligning of the sequences of each of the multiple fragments of cDNA to the reference TCR sequence database may be carried out, for example, using the Burrows- Wheeler Aligner (BWA) software package (//bio-bwa.sourceforge.net/), described in Li et al., Bioinformatics, 25: 1754-60 (2009) and Li et al, Bioinformatics, 26(5): 589-95 (2010).
  • BWA Burrows- Wheeler Aligner
  • the method further comprises identifying TCR complementarity determining region 3 (CDR3) sequences in the multiple fragments of cDNA containing the identified TCR alpha chain V segment sequences and in the multiple fragments of cDNA containing the identified TCR beta chain V segment sequences.
  • the CDR3 region sequence may be identified in any suitable manner.
  • identifying TCR CDR3 sequences is carried out by identifying cDNA sequences which encode conserved amino acid residues positioned near the C-terminus of the amino acid sequence which is encoded by the V segment of the alpha and beta chains.
  • identifying a TCR CDR3 sequence may be carried out by identifying any cDNA sequence(s) which encodes the amino acid sequence motif of
  • each of X1-X9 is any naturally occurring amino acid
  • each of X10-X21 is no amino acid or is any naturally occurring amino acid
  • X22 is phenylalanine or tryptophan.
  • the amino acid sequence motif of SEQ ID NO: 5 is a conserved amino acid sequence motif positioned near the C-terminus of the amino acid sequence encoded by the V segment.
  • the method further comprises identifying the TCR alpha chain constant (C) region sequence of the highest number of multiple fragments of cDNA collected and the TCR beta chain C region sequence of the highest number of multiple fragments of cDNA collected.
  • the method further comprises identifying the TCR alpha chain C region sequence of the second highest number of multiple fragments of cDNA collected.
  • a TCR alpha chain has one possible constant region amino acid sequence.
  • a TCR beta chain has one of two possible constant region amino acid sequences.
  • the method further comprises counting the number of multiple fragments of cDNA which share the same alpha chain CDR3 amino acid sequence and the number of multiple fragments of cDNA which share the same beta chain CDR3 amino acid sequence.
  • the method further comprises collecting the highest number of multiple fragments of cDNA which encode the same alpha chain CDR3 sequence, the highest number of multiple fragments of cDNA which encode the same beta chain CDR3 sequence and, optionally, the second highest number of multiple fragments of cDNA which encode the same alpha chain CDR3 sequence to identify TCR alpha and beta chain CDR3 sequences.
  • the alpha chain CDR3 sequence encoded by the second highest number of multiple fragments of cDNA is different from the alpha chain CDR3 sequence encoded by the highest number of multiple fragments of cDNA.
  • the CDR3 sequences identified may include the beta chain CDR3 sequence and the alpha chain CDR3 sequence of the TCR having antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation and, optionally, an additional alpha chain CDR3 sequence expressed by the T cell but which does not pair with the beta chain CDR3 sequence to form the TCR having antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation. It is estimated that about a third of mature T cells may express two TCR alpha chains. Only one of the expressed alpha chains will pair with the expressed TCR beta chain to provide a TCR which has antigenic specificity for the amino acid sequence encoded by the cancer-specific mutation.
  • the method further comprises identifying the TCR alpha chain V segment sequence of the highest number of multiple fragments of cDNA collected, the TCR beta chain V segment sequence of the highest number of multiple fragments of cDNA collected, and, optionally, the TCR alpha chain V segment sequence of the second highest number of multiple fragments of cDNA collected to identify TCR alpha and beta chain V segment sequences.
  • the number of multiple fragments of cDNA which encode the CDR3 sequence of the dominant TCR expressed by a single, activated T cell will outnumber the number of fragments of cDNA which encode any other TCR CDR3 sequence which may be present due to contamination by a factor of about 10 to about 100.
  • the source of the contamination may be nearby single-cell samples or unknown sources.
  • the dominant TCR expressed by the single T cell which expressed one or more T cell activation markers in response to co-culture with APCs that present the mutated amino acid sequence, is a TCR which has antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.
  • the method further comprises assembling one or more nucleotide sequences encoding a TCR alpha chain comprising the identified TCR alpha chain V segment sequence identified and the collected TCR alpha chain CDR3 sequence and a TCR beta chain comprising the identified TCR beta chain V segment sequence and the collected TCR beta chain CDR3 sequence.
  • the various multiple fragments of cDNA which encode the same CDR3 sequence may be of various lengths and may overlap with one another. By aligning the various multiple fragments of cDNA which encode the same alpha chain CDR3 sequence of various lengths with one another, the sequence of the entire V segment, J segment, and, optionally, the constant region, of the dominant TCR alpha chain can be determined.
  • the sequence of the entire V segment, J segment, D segment, and, optionally, the constant region, of the dominant TCR beta chain can be determined.
  • a nucleotide sequence encoding the entire V segment, J segment, and, optionally, the constant region, of the dominant TCR alpha chain and a nucleotide sequence encoding the entire V segment, J segment, D segment, and, optionally, the constant region, of the dominant TCR beta chain can be assembled using routine techniques. Isolated paired TCR alpha and beta chain sequences, or an antigen-binding portion thereof, may be produced.
  • the assembling of one or more nucleotide sequences comprises assembling a TCR alpha chain comprising the TCR alpha chain V segment sequence identified in the sample, the TCR alpha chain C region sequence identified in the sample, and the TCR alpha chain CDR3 sequence collected and assembling a TCR beta chain comprising the TCR beta chain V segment sequence identified in the sample, the TCR beta chain C region sequence identified in the sample, and the TCR beta chain CDR3 sequence collected.
  • the nucleotide sequences assembled may comprise an endogenous C region sequence.
  • the assembling of one or more nucleotide sequences comprises assembling a TCR alpha chain comprising the TCR alpha chain V segment sequence identified in the sample, an exogenous TCR alpha chain C region sequence, and the TCR alpha chain CDR3 sequence collected and assembling a TCR beta chain comprising the TCR beta chain V segment sequence identified in the sample, an exogenous TCR beta chain C region sequence, and the TCR beta chain CDR3 sequence collected.
  • An exogenous C region sequence is a C region sequence that is not native to (not naturally-occurring on) the T cell.
  • the isolated paired TCR alpha and beta chain sequence, or an antigen-binding portion thereof, produced by the method may be a chimeric or hybrid TCR comprised of amino acid sequences derived from TCRs from two different mammalian species.
  • the TCR can comprise a variable region derived from a human TCR and a constant region of a mouse TCR such that the TCR is "murinized.”
  • Methods of making chimeric or hybrid TCRs are described in, for example, Cohen et al., Cancer Res., 66: 8878-8886 (2006); Cohen et al., Cancer Res., 67: 3898-3903 (2007); and Haga-Friedman et al., J. Immunol, 188: 5538-5546 (2012)).
  • a single T cell typically expresses one TCR beta chain and one or two TCR alpha chains.
  • the presence of more than one TCR beta chain in a single sample may be the result of imperfect sorting of the T cells into separate T cell samples. Imperfect sorting may result in two or more T cells inadvertently being included in one sample. If a single sample is found to express more than one TCR beta chain, that sample may be eliminated from subsequent analysis.
  • T cells may express two TCR alpha chains. Only one of the expressed alpha chains will pair with the expressed TCR beta chain to provide a TCR which has antigenic specificity for the amino acid sequence encoded by the cancer-specific mutation.
  • the method may comprise assembling a first nucleotide sequence encoding a first TCR alpha chain comprising the first TCR alpha chain V segment sequence of the highest number of multiple fragments of cDNA identified as described herein and the first TCR alpha chain CDR3 sequence collected as described herein and a TCR beta chain comprising the TCR beta chain V segment sequence identified as described herein and the TCR beta chain CDR3 sequence collected as described herein.
  • the method may optionally further comprise assembling a second one or more nucleotide sequences encoding: a second TCR alpha chain comprising the TCR alpha chain V segment sequence of the second highest number of multiple fragments of cDNA identified and the TCR alpha chain CDR3 sequence of the second highest number of multiple fragments of cDNA collected and the TCR beta chain comprising the TCR beta chain V segment sequence identified and the TCR beta chain CDR3 sequence collected.
  • the method may further comprise independently introducing the first and second nucleotide sequences into first and second populations of host cells, respectively, and independently co-culturing the first and second populations of host cells with APCs that present the mutated amino acid sequence encoded by a cancer-specific mutation.
  • the method may further comprise selecting the population of host cells that (a) were co-cultured with the APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence.
  • the co-cultured population of host cells that has antigenic specificity for the mutated amino acid sequence will express the TCR alpha chain which, together with the TCR beta chain, provides the desired specificity.
  • Cells which have antigenic specificity for the mutated amino acid sequence may be identified by any suitable means known in the art.
  • cells which have antigenic specificity for the mutated amino acid sequence may be identified on the basis of expression of one or more T cell activation markers and/or one or more cytokines, as described in, for example, WO 2016/053338 and WO 2016/053339.
  • the T cell activation markers may be as described herein with respect to other aspects of the invention.
  • the cytokine may comprise any cytokine the secretion of which by a T cell is characteristic of T cell activation (e.g., a TCR expressed by the T cells specifically binding to and
  • Non-limiting examples of cytokines include IFN- ⁇ , IL-2, granzyme B, and tumor necrosis factor alpha (TNF-a), granulocyte/monocyte colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.
  • an embodiment of the invention provides a method of automatically identifying the TCR alpha and beta chain V segment sequences and CDR3 sequences of a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation.
  • Figure 6 is a block diagram of a system 100 in accordance with certain
  • the system 100 may include one or more sequencer computer device(s) 101 , a user computing device 103, and a network connection 102 between the user computing device 103 and the sequencer computing device 101.
  • the sequencer computing device 101 may be any system which is capable of sequencing the mRNA from each separate single T cell sample. Examples of sequencer computing devices 101 may include any of the NGS technologies and platforms described herein with respect to other aspects of the invention.
  • the user computing device 101 can be any type of communication device that supports network communication, including a personal computer, a laptop computer, or a personal digital assistant (PDA), etc.
  • the user computing device 101 can support multiple types of networks.
  • the user computing device 101 may have wired or wireless network connectivity using IP (Internet Protocol) or may have mobile network connectivity allowing over cellular and data networks.
  • IP Internet Protocol
  • user computing device 103 is used to capture the sequences of each of the multiple fragments of cDNA provided by the sequencer computing device 101.
  • the sequences may be transmitted over a network connection 102.
  • An example of a network connection 102 is shared disk space.
  • FIG. 7 is a block diagram of basic functional components for a computing device 103 according to some aspects of the invention.
  • the computing device 103 includes one or more processors 202, memory 204, network interfaces 206, storage devices 208, power source 210, one or more output devices 212, one or more input devices 214, and software modules - operating system 216 and a sequence application 218 - stored in memory 204.
  • the software modules are provided as being contained in memory 204, but in certain embodiments, the software modules are contained in storage devices 208 or a combination of memory 204 and storage devices 208.
  • Each of the components including the processor 202, memory 204, network interfaces 206, storage devices 208, power source 210, output devices 212, input devices 214, operating system 216, and the sequence application 218, is interconnected physically,
  • processor 202 is configured to implement functionality and/or process instructions for execution within client device 103.
  • processor 202 executes instructions stored in memory 204 or instructions stored on a storage device 208.
  • Memory 204 which may be a non-transient, computer-readable storage medium, is configured to store information within client device 103 during operation.
  • memory 204 includes a temporary memory, an area for information not to be maintained when the client device 103 is turned off. Examples of such temporary memory include volatile memories such as random access memories (RAM), dynamic random access memories (DRAM), and static random access memories (SRAM).
  • RAM random access memories
  • DRAM dynamic random access memories
  • SRAM static random access memories
  • Storage device 208 also includes one or more non-transient computer-readable storage media.
  • the storage device 208 is generally configured to store larger amounts of information than memory 204.
  • the storage device 208 may further be configured for long- term storage of information.
  • the storage device 208 includes nonvolatile storage elements.
  • Non-limiting examples of non-volatile storage elements include magnetic hard discs, optical discs, floppy discs, flash memories, or forms of electrically programmable memories (EPROM) or electrically erasable and programmable (EEPROM) memories.
  • Network interface 206 may be a network interface card, such as an Ethernet card, an optical transceiver, a radio frequency transceiver, or any other type of device that can send and receive information.
  • network interfaces include Bluetooth®, 3G and Wi-Fi radios in client computing devices, and Universal Serial Bus (USB).
  • User computing device 103 includes one or more power sources 210 to provide power to the device.
  • power source 210 include single-use power sources, rechargeable power sources, and/or power sources developed from nickel-cadmium, lithium-ion, or other suitable material.
  • One or more output devices 212 are also included in user computing device 103.
  • Output devices 212 are configured to provide output to a user using tactile, audio, and/or video stimuli.
  • Output device 212 may include a display screen (part of the presence-sensitive screen), a sound card, a video graphics adapter card, or any other type of device for converting a signal into an appropriate form understandable to humans or machines.
  • output device 212 includes a speaker such as headphones, a cathode ray tube (CRT) monitor, a liquid crystal display (LCD), or any other type of device that can generate intelligible output to a user.
  • a speaker such as headphones, a cathode ray tube (CRT) monitor, a liquid crystal display (LCD), or any other type of device that can generate intelligible output to a user.
  • CTR cathode ray tube
  • LCD liquid crystal display
  • the user computing device includes one or more input devices 214.
  • Input devices 214 are configured to receive input from a user or a surrounding environment of the user through tactile, audio, and/or video feedback.
  • Non-limiting examples of input device 214 include a photo and video camera, presence-sensitive screen, a mouse, a keyboard, a voice responsive system, microphone or any other type of input device.
  • a presence-sensitive screen includes a touch-sensitive screen.
  • the client device 103 includes an operating system 216.
  • the operating system 216 controls operations of the components of the client device 103.
  • the operating system 216 facilitates the interaction of the processor(s) 202, memory 204, network interface 206, storage device(s) 208, input device 214, output device 212, and power source 210.
  • the user computing device may use sequence application 218 to capture the sequences of the multiple fragments of cDNA of the single T cell(s) identified to express one or more T cell activation markers following co-culture with APCs that present the mutated amino acid sequence.
  • the sequence application 218 may interface with and receive inputs from a sequencer computing device.
  • the user may download the sequences of the multiple fragments of cDNA of a single identified T cell from the sequencer computing device 101 to a removable disk such as, for example, a USB flash drive.
  • the user computing device may obtain the sequences of the multiple fragments of cDNA of a single identified T cell from the removable disk.
  • the user computing device 103 may include software stored in a memory and executed by a processor to identify the TCR alpha and beta chain V segment sequences and CDR3 sequences of a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, as described herein with respect to other aspects of the invention.
  • FIG 8 is a flow diagram of method steps for automatically identifying the TCR alpha and beta chain V segment sequences and CDR3 sequences of a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation.
  • the method 400 begins at step 402, where a user computing device 103 receives the sequences of the multiple fragments of cDNA of the single T cell(s) which was/were identified to express one or more T cell activation markers following co-culture with APCs that present the mutated amino acid sequence encoded by the cancer-specific mutation.
  • the method may comprise receiving the sequences of the multiple fragments of cDNA at the computing device over an electronic network or via a removable disk (e.g., USB drive).
  • the user computing device 103 performs computerized alignment of the sequences of each of the multiple fragments of cDNA to a reference TCR sequence database to identify TCR alpha chain V segment sequences and TCR beta chain V segment sequences of the multiple fragments of cDNA of the single T cell identified to express one or more T cell activation markers following co-culture with APCs that present the mutated amino acid sequence encoded by the cancer-specific mutation.
  • the user computing device 103 performs computerized identification of TCR CDR3 sequences in the multiple fragments of cDNA containing the identified TCR alpha chain V segment sequences and in the multiple fragments of cDNA containing the identified TCR beta chain V segment sequences. [0081] At step 405, the user computing device 103 performs computerized counting of the number of multiple fragments of cDNA which share the same alpha chain CDR3 amino acid sequence and the number of multiple fragments of cDNA which share the same beta chain CDR3 amino acid sequence.
  • the user computing device performs computerized collecting of the highest number of multiple fragments of cDNA which encode the same alpha chain CDR3 sequence, the highest number of multiple fragments of cDNA which encode the same beta chain CDR3 sequence and, optionally, the second highest number of multiple fragments of cDNA which encode the same alpha chain CDR3 sequence, wherein the alpha chain CDR3 sequence encoded by the second highest number of multiple fragments of cDNA is different from the alpha chain CDR3 sequence encoded by the highest number of multiple fragments of cDNA to identify the TCR alpha and beta chain CDR3 sequences.
  • the user computing device 103 performs computerized identification of the TCR alpha chain V segment sequence of the highest number of multiple fragments of cDNA collected, the TCR beta chain V segment sequence of the highest number of multiple fragments of cDNA collected and, optionally, the TCR alpha chain V segment sequence of the second highest number of multiple fragments of cDNA collected to identify the TCR alpha and beta chain V segment sequences.
  • Another embodiment of the invention provides a pair of TCR alpha and beta chain sequences, or an antigen-binding portion thereof, isolated according to any of the methods described herein with respect to other aspects of the invention.
  • An embodiment of the invention provides an isolated TCR comprising two polypeptides (i.e., polypeptide chains), such as an alpha (a) chain of a TCR and a beta ( ⁇ ) chain of a TCR.
  • polypeptides of the inventive isolated pairs of TCR alpha and beta chain sequences can comprise any amino acid sequence, provided that the TCR, or the antigen-binding portion thereof, has antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.
  • the antigen-binding portion of the isolated pair of TCR alpha and beta chain sequences refers to any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the antigen-binding portion specifically binds to the mutated amino acid sequence encoded by the cancer-specific mutation as described herein with respect to other aspects of the invention.
  • the term "antigen-binding portion” refers to any part or fragment of the TCR isolated by the inventive methods, which part or fragment retains the biological activity of the TCR of which it is a part (the parent TCR).
  • Antigen- binding portions encompass, for example, those parts of a TCR that retain the ability to specifically bind to the mutated amino acid sequence, or detect, treat, or prevent cancer, to a similar extent, the same extent, or to a higher extent, as compared to the parent TCR.
  • the antigen-binding portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent TCR.
  • the antigen-binding portion can comprise an antigen-binding portion of either or both of the a and ⁇ chains of the TCR isolated by the inventive methods, such as a portion comprising one or more of the complementarity determining region (CDR)l, CDR2, and CDR3 of the variable region(s) of the a chain and/or ⁇ chain of the TCR isolated by the inventive methods.
  • CDR complementarity determining region
  • the antigen-binding portion can comprise the amino acid sequence of the CDR1 of the a chain (CDRla), the CDR2 of the a chain (CDR2a), the CDR3 of the a chain (CDR3a), the CDR1 of the ⁇ chain (CDRip), the CDR2 of the ⁇ chain (CDR2 ⁇ ), the CDR3 of the ⁇ chain (CDR3 ⁇ ), or any combination thereof.
  • the antigen-binding portion comprises the amino acid sequences of CDRla, CDR2a, and CDR3a; the amino acid sequences of CDR ⁇ , CDR2 ⁇ , and CDR3 ⁇ ; or the amino acid sequences of all of CDRla, CDR2a, CDR3a, CDR ⁇ , CDR2 ⁇ , and ⁇ 3 ⁇ of the TCR isolated by the inventive methods.
  • the antigen-binding portion can comprise, for instance, the variable region of the TCR isolated by the inventive methods comprising a combination of the CDR regions set forth above, for example, all six CDR regions set forth above.
  • the antigen-binding portion can comprise the amino acid sequence of the variable region of the a chain (Va), the amino acid sequence of the variable region of the ⁇ chain ( ⁇ ), or the amino acid sequences of both of the Va and ⁇ of the TCR isolated by the inventive methods.
  • the antigen-binding portion may comprise a combination of a variable region and a constant region.
  • the antigen-binding portion can comprise the entire length of the a or ⁇ chain, or both of the a and ⁇ chains, of the TCR isolated by the inventive methods.
  • the isolated paired TCR alpha and beta chain sequences, or the antigen-binding portion thereof, isolated by the inventive methods may be useful for preparing cells for adoptive cell therapies.
  • another embodiment of the method provides a method of preparing a population of cells that express paired TCR alpha and beta chain sequences, or an antigen-binding portion thereof.
  • the method may comprise isolating paired TCR alpha and beta chain sequences, or an antigen-binding portion thereof, according to any of the methods described herein with respect to other aspects of the invention.
  • the method may further comprise introducing the nucleotide sequence encoding the isolated paired TCR alpha and beta chain sequences, or the antigen-binding portion thereof, into host cells to obtain cells that express the paired TCR alpha and beta chain sequences, or the antigen-binding portion thereof.
  • the method may comprise cloning the nucleotide sequence that encodes the isolated paired TCR alpha and beta chain sequences, or the antigen-binding portion thereof, into a recombinant expression vector using established molecular cloning techniques as described in, e.g., Green et al. (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed. (2012).
  • the term "recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed by the cell.
  • the vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring.
  • the recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vectors can comprise naturally-occurring, non-naturally-occurring internucleotide linkages, or both types of linkages.
  • the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.
  • the recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell.
  • Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • the vector can be selected from the group consisting of transposon/transposase, the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series
  • Bacteriophage vectors such as GTIO, GTl 1 , ZapII (Stratagene), ⁇ 4, and ⁇ 149, also can be used.
  • plant expression vectors include pBIOl , pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).
  • the recombinant expression vector is a viral vector, e.g., a retroviral vector.
  • nucleotide sequence e.g., a recombinant expression vector
  • introducing the nucleotide sequence encoding the isolated paired TCR alpha and beta chain sequences, or the antigen-binding portion thereof, into host cells
  • Non-limiting examples of techniques that are useful for introducing a nucleotide sequence into host cells include transformation, transduction, transfection, and electroporation.
  • the method comprises introducing the nucleotide sequence encoding the isolated paired TCR alpha and beta chain sequences, or the antigen-binding portion thereof, into host cells that are autologous to the patient who provided the biological sample.
  • the TCRs, or the antigen-binding portions thereof, identified and isolated by the inventive methods may be personalized to each patient.
  • the inventive methods may identify and isolate TCRs, or the antigen-binding portions thereof, that have antigenic specificity against a mutated amino acid sequence that is encoded by a recurrent (also referred to as "hot-spot") cancer-specific mutation.
  • the method may comprise introducing the nucleotide sequence encoding the isolated paired TCR alpha and beta chain sequences, or the antigen-binding portion thereof, into host cells that are allogeneic to the patient.
  • the method may comprise introducing the nucleotide sequence encoding the isolated paired TCR alpha and beta chain sequences, or the antigen-binding portion thereof, into the host cells of another patient whose tumors express the same mutation in the context of the same MHC molecule as the patient who originally expressed the TCR.
  • the host cells are peripheral blood
  • the PBMC may include T cells.
  • T cells can be obtained from numerous sources in the patient, including but not limited to tumor, blood, bone marrow, lymph node, the thymus, or other tissues or fluids.
  • the T cells can include any type of T cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells, e.g., Thl and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells (e.g., tumor infiltrating lymphocytes (TIL)), peripheral blood T cells, memory T cells, naive T cells, and the like.
  • CD4+/CD8+ double positive T cells CD4+ helper T cells, e.g., Thl and Th2 cells
  • CD8+ T cells e.g., cytotoxic T cells
  • tumor infiltrating cells e.g., tumor infiltrating lymphocytes (TIL)
  • the T cells may be CD8+ T cells, CD4+ T cells, or both CD4+ and CD8+ T cells. [0095] Without being bound to a particular theory or mechanism, it is believed that less differentiated, "younger” T cells may be associated with any one or more of greater in vivo persistence, proliferation, and antitumor activity as compared to more differentiated, "older" T cells.
  • the inventive methods may, advantageously, identify and isolate paired TCR alpha and beta chain sequences, or an antigen-binding portion thereof, that has antigenic specificity for the mutated amino acid sequence and introduce the paired TCR alpha and beta chain sequences, or an antigen-binding portion thereof, into "younger" T cells that may provide any one or more of greater in vivo persistence, proliferation, and antitumor activity as compared to "older" T cells (e.g., effector cells in a patient's tumor) from which the TCR, or the antigen-binding portion thereof, may have been isolated.
  • "younger" T cells e.g., effector cells in a patient's tumor
  • the method further comprises expanding the numbers of host cells into which the TCR, or the antigen-binding portion thereof has been introduced. Expansion of the numbers of cells can be accomplished by any of a number of methods as are known in the art as described in, for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; U.S. Patent Application Publication No. 2012/0244133; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128: 189-201 (1990). In an embodiment, expansion of the numbers of cells is carried out by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMC (e.g., irradiated allogeneic PBMC).
  • PBMC e.g., irradiated allogeneic PBMC
  • Another embodiment of the invention provides an isolated population of cells prepared according to any of the methods described herein with respect to other aspects of the invention.
  • the population of cells can be a heterogeneous population comprising the host cells expressing the isolated TCR, or the antigen-binding portion thereof, in addition to at least one other cell, e.g., a host cell (e.g., a PBMC), which does not express the isolated TCR, or the antigen-binding portion thereof, or a cell other than a T cell, e.g., a B cell, a
  • a host cell e.g., a PBMC
  • a cell other than a T cell e.g., a B cell, a
  • the population of cells can be a substantially homogeneous population, in which the population comprises mainly of (e.g., consisting essentially of) host cells expressing the isolated TCR, or the antigen-binding portion thereof.
  • the population also can be a clonal population of cells, in which all cells of the population are clones of a single PBMC expressing the isolated TCR, or the antigen-binding portion thereof, such that all cells of the population express the isolated TCR, or the antigen-binding portion thereof.
  • the population of cells is a clonal population comprising host cells expressing the isolated TCR, or the antigen-binding portion thereof, as described herein.
  • the inventive methods may, advantageously, provide a population of cells that comprises a high proportion of host cells cells that express the isolated TCR and have antigenic specificity for the mutated amino acid sequence.
  • about 1 % to about 100% for example, about 1 %, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, or a range defined by any two of the foregoing values, of the population of cells comprises host cells that express the isolated TCR and have antigenic specificity for the mutated amino acid sequence.
  • populations of cells that comprise a high proportion of host cells that express the isolated TCR and have antigenic specificity for the mutated amino acid sequence have a lower proportion of irrelevant cells that may hinder the function of the host cells, e.g., the ability of the host cells to target the destruction of cancer cells and/or treat or prevent cancer.
  • inventive TCRs, or the antigen-binding portions thereof, and populations of cells can be formulated into a composition, such as a pharmaceutical composition.
  • the invention provides a pharmaceutical composition comprising any of the inventive TCRs, or the antigen-binding portions thereof, or populations of cells and a pharmaceutically acceptable carrier.
  • inventive pharmaceutical composition can comprise an inventive TCR, or an antigen-binding portion thereof, or population of cells in combination with another pharmaceutically active agent(s) or drug(s), such as a chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorabicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • chemotherapeutic agents e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorabicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • the carrier is a pharmaceutically acceptable carrier.
  • the carrier can be any of those conventionally used for the particular inventive TCR, or the antigen-binding portion thereof, or population of cells under consideration.
  • Such pharmaceutically acceptable earners are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable earner be one which has no detrimental side effects or toxicity under the conditions of use.
  • the choice of carrier will be determined in part by the particular inventive TCR, the antigen-binding portion thereof, or population of cells, as well as by the particular method used to administer the inventive TCR, the antigen-binding portion thereof, or population of cells. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. Suitable formulations may include any of those for oral, parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, or
  • More than one route can be used to administer the inventive TCR or population of cells, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
  • the inventive TCR, the antigen-binding portion thereof, or population of cells is administered by injection, e.g., intravenously.
  • the pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate.
  • the pharmaceutically acceptable carrier is supplemented with human serum albumin.
  • inventive TCRs, the antigen-binding portions thereof, populations of cells, and pharmaceutical compositions can be used in methods of treating or preventing cancer.
  • inventive TCRs, or the antigen-binding portions thereof are believed to bind specifically to a mutated amino acid sequence encoded by a cancer-specific mutation, such that the TCR, or the antigen-binding portion thereof, when expressed by a cell, is able to mediate an immune response against a target cell expressing the mutated amino acid sequence.
  • an embodiment of the invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the inventive pharmaceutical compositions, isolated pairs of TCR alpha and beta chain sequences, antigen-binding portions thereof, or populations of cells described herein, in an amount effective to treat or prevent cancer in the mammal.
  • Another embodiment of the invention provides any of the inventive TCRs or antigen-binding portions thereof, populations of cells, or pharmaceutical compositions described herein with respect to other aspects of the invention for use in treating or preventing cancer in a mammal.
  • the terms "treat,” and “prevent” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect.
  • the inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.
  • treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented.
  • treatment or prevention can include promoting the regression of a tumor.
  • prevention can encompass delaying the onset of the cancer, or a symptom or condition thereof.
  • the amount or dose of the inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the mammal over a reasonable time frame.
  • the dose of the inventive TCR, the antigen- binding portion thereof, population of cells, or pharmaceutical composition should be sufficient to bind to a mutated amino acid sequence encoded by a cancer-specific mutation, or detect, treat or prevent cancer in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer.
  • the dose will be determined by the efficacy of the particular inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition administered and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated.
  • an assay which comprises comparing the extent to which target cells are lysed or IFN- ⁇ is secreted by T cells expressing the inventive TCR, or the antigen- binding portion thereof, upon administration of a given dose of such T cells to a mammal among a set of mammals of which is each given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal.
  • the extent to which target cells are lysed or IFN- ⁇ is secreted upon administration of a certain dose can be assayed by methods known in the art.
  • the dose of the inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition.
  • the attending physician will decide the dosage of the inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition to be administered, route of administration, and the severity of the condition being treated.
  • the number of cells administered per infusion may vary, for example, in the range of one million to 100 billion cells; however, amounts below or above this exemplary range are within the scope of the invention.
  • the daily dose of inventive host cells can be about 1 million to about 150 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, about 60 billion cells, about 80 billion cells, about 100 billion cells, about 120 billion cells, about 130 billion cells, about 150 billion cells, or a range defined by any two of the foregoing values), preferably about 10 million to about 130 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, about 100 billion cells, about
  • the cells can be cells that are allogeneic or autologous to the mammal.
  • the cells are autologous to the mammal.
  • Another embodiment of the invention provides any of the inventive TCRs, the antigen-binding portions thereof, isolated population of cells, or pharmaceutical compositions described herein for use in treating or preventing cancer in a mammal.
  • the cancer may, advantageously, be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, cholangiocarcinoma, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, uterine cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma,
  • the mammal referred to in the inventive methods can be any mammal.
  • the term "mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). Preferably, the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses).
  • the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • a more preferred mammal is the human.
  • the mammal is the patient expressing the cancer-specific mutation.
  • Tandem minigene (TMG) libraries encompassing the nonsynonymous mutationswere synthesized.
  • DCs dendritic cells
  • TMG Tandem minigene
  • RNA-seq A method of identifying neoantigen-specific TCR sequences from single-cell RNA sequencing (RNA-seq) data is summarized in the schematic shown in Figure 2.
  • 1 X 10 6 TILs were co-cultured with 1 X 10 6 TMG-pulsed DCs for four (4) hours (hr).
  • the T cells were re-suspended and washed extensively. Then the T cells were subjected to single-cell sorting and RNA-seq sample preparation according to the manufacturers' instructions (Fluidigm (San Francisco, CA) and Clontech (Mountain View, CA)).
  • RNA-seq samples were barcoded using the NEXTERA XT DNA Library Preparation Kit (lllumina (San Diego, CA)) and were then sequenced using the ILLUMINA MISEQ system using the reagent kit V3 (2 x 250 base pairs (b.p.)).
  • RNA-seq data were aligned by Burrows-Wheeler Aligner (BWA) (Li et al., Bioinformatics, 25: 1754-60 (2009); Li et al., Bioinformatics, 26(5): 589-95 (2010)) using TCRa/ ⁇ variable (V) region sequences from the international immunogenetics information system (IMGT) database (Lefranc et al., Nucleic Acids Res., 43: D413-422 (2015)). CDR3 region sequences were identified based on the conservative amino acid residues (C...F/W) near the C-terminus of the V region, analyzed and reported by software.
  • BWA Burrows-Wheeler Aligner
  • TCRs with non-productive (out of frame) sequences were removed from the analysis. Additionally, some samples may have contained more than one T cell due to the imperfect sorting mechanism of the FLUIDIGM CI single-cell mRNA sequencing system. As a result, samples with more than one TCRp were eliminated from the subsequent analysis.
  • RNA-seq data were aligned against the sequence of IFN- ⁇ , IL-2 or other potential T cell activation markers. Aligned TCR fragments that associated with high IFN- ⁇ single- cells were extracted, and TCR V, CDR3 and constant (C) regions were identified. To assemble paired full-length TCR sequences, incomplete 5' V region sequences were assembled with the identified human full-length TCR V region sequences from the IMGT database.
  • MSGV8 retroviral expression vector (RAKRSGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO: 1), were synthesized and cloned into a MSGV8 retroviral expression vector (Wargo et al., Cancer Immunol. Immunother., 58: 383-394 (2009)).
  • MSGV8-TCR plasmid (1.5 ⁇ g) and 0.75 ⁇ g of VSV-G (RD1 14) plasmid were co-transfected into 1 X 10 6 293GP cells in each 6-well using LIPOFECTAMINE 2000 Transfection Reagent (Thermo Fisher Scientific). After 48 hr, the supernatant was harvested and spun at 3000 revolutions per minute (rpm) for 10 minutes (min) to remove debris. The retrovirus supernatant was loaded on RETRONECTIN reagent (Takara, Otsu, Japan) coated 6-well plates by centrifugation at 2000 g for 2 hr.
  • PBMCs 1 X 10 6 /mL PBMCs from healthy donors were stimulated with 50 ng/mL anti-CD3 mAb OKT3 and 300 IU/mL IL-2 in AIM V medium containing 5% human serum. After 2 days, stimulated cells were harvested and re-suspended in the same medium without O T3. Stimulated PBMCs were added to each retro virus-loaded well at 2 X 10 6 cells/well and spun at 1000 g for 10 min. Plates were incubated overnight at 37 °C. The next day, the PBMCs were transferred to new retrovirus-loaded wells, and the transduction procedure was repeated. TCR-transduced T cells were continuously cultured in AIM V medium with 300 IU/mL IL-2 and 5% human serum for 5 more days before experiments.
  • This example demonstrates a method of isolating the paired alpha and beta chain sequences of a neoantigen-specific TCR from the TIL 4090 culture.
  • TIL 4090 cultures were grown from a metastatic lung lesion resected from a patient with colorectal cancer.
  • One of the cultures, TIL 4090 F7 recognized TMG-5 based on the results of TMG library screening.
  • TIL 4090 F7 cells were co-cultured with TMG-5-pulsed autologous DCs for 4 hr and subjected to single-cell RNA-seq analysis.
  • sequence reads in the single-cell RNA-seq data two single cells expressed high percentages of IFN- ⁇ reads (6.42% and 12.25% of the total Rl reads) (Fig. 3 A).
  • Example 1 T cells transduced with the TCR alpha and beta chain sequences isolated in Example 1 specifically recognize a neoantigen expressed by the cancer of the patient of Example 1.
  • TCR isolated from TIL 4090 F7 the full-length TCRa and TCRp sequences with modified mouse constant regions, linked by a furin SGSGP2A linker, were synthesized and cloned into a MSGV8 retroviral expression vector.
  • Peripheral blood T cells were transduced with the 4090 TCR and co-cultured with TMG-5-pulsed 4090 DCs overnight. Based on the secretion of IFN- ⁇ by T cells, 4090 TCR-transduced T cells recognized TMG-5-pulsed DCs, but not DCs pulsed with irrelevant TMG (Fig. 3C).
  • TMG-5 contained a total of 12 minigenes. 25-mer mutated long-peptides corresponding to each minigene were synthesized and pulsed onto 4090 DCs for 24 hr. After washing, peptide-pulsed DCs were co-cultured with the 4090 TCR-transduced T cells overnight. The 4090 TCR-transduced T cells only reacted to the DCs pulsed with mutated USP8 (ubiquitin specific peptidase 8) peptide WAKFLDPITGTFHYYHSPTNTVHMY (R>H) (SEQ ID NO: 2), suggesting that the 4090 TCR recognized mutated USP8 ( Figure 3D).
  • mutated USP8 ubiquitin specific peptidase 8
  • This example demonstrates a method of isolating the paired alpha and beta chain sequences of a neoantigen-specific TCR from the TIL 4095 culture.
  • TIL 4095 cultures were grown from metastatic lung lesions resected from a patient with colorectal cancer.
  • TIL 4095 F5 recognized TMG-1 based on the results of TMG library screening.
  • TIL 4095 F5 cells were co- cultured with TMG-1 -pulsed autologous DCs for 4 hr and subjected to single-cell RNA-seq analysis. All single-cells with high levels of IFN- ⁇ reads (0.79% ⁇ 3.74%) were identified (Fig. 4A). These T cells all contained exactly the same TCRa/ ⁇ variable and CDR3 sequences (Table 2). Only one single-cell expressed detectable IL-2 reads (0.03%) (Fig. 4B), and this single cell co-expressed IFN- ⁇ at a high level (1.07%) (Fig. 4C).
  • Example 3 T cells transduced with the TCR alpha and beta chain sequences isolated in Example 3 specifically recognize a neoantigen expressed by the cancer of the patient of Example 3.
  • TCR isolated from TIL 4095 F5 of Example 3 the full-length TCRa and TCR sequences with modified mouse constant regions were synthesized and cloned into a MSGV8 retroviral expression vector, and then transduced into donor T cells.
  • a TCR which recognizes a mutated KRAS(G12D) peptide in an HLA- C0802-restricted manner was identified (Tran et al., Science, 350: 1387-1390 (2015)).
  • This example demonstrates a method of isolating the paired alpha and beta chain sequences of a neoantigen-specific TCR from the TIL 41 12 culture.
  • TIL 41 12 cultures were grown from a metastatic liver lesion resected from a patient with cholangiocarcinoma.
  • TIL 41 12 F5 was found to recognize TMG-9 based on the results of TMG library screening.
  • TIL 4112 F5 cells were co-cultured with TMG-9-pulsed autologous DCs for 4 hr and subjected to single-cell RNA-seq analysis. Twenty-two (22) single-cells with high levels of IFN- ⁇ reads (>2%) contained exactly the same TCR sequences (Fig. 5A, Fig. 5C, and Table 3).
  • TMG-9 was submitted to the Immune Epitope Database (IEDB) And Analysis Resource website (iedb.org) and Center for Biological Sequence (CBS) Analysis NetMHC website (cbs.dtu.dk/services/NetMHC/) to predict peptides with high affinity to the 6 HLAs of patient 4112.
  • IEDB Immune Epitope Database
  • CBS Center for Biological Sequence
  • NetMHC NetMHC website
  • This example demonstrates a method of isolating the paired alpha and beta chain sequences of a neoantigen-specific TCR from the TIL 4171 culture.
  • TIL 4171 cultures were grown from a metastatic lung lesion resected from a patient with colorectal cancer. 128 long-peptides (25-mer) were synthesized, and each peptide contained a nonsynonymous mutation flanked on both sides by 12 normal amino acids. TIL 4171 cultures were screened against the peptide library, and one of the cultures, TIL4171F6, recognized peptide pool 3 (PP-3) ( Figure 9A). TIL4171F6 cells were then co- cultured with PP-3-pulsed autologous DCs for 4 hr, and subjected to single-cell RNA-seq analysis. Expression of IFN- ⁇ and IL-2 was measured (Figs. 9B-9D).
  • V gene segments have been found to be rearranged to AJ joining gene segments for TCRa, and to be rearranged to DD diversity gene segments and DJ joining gene segments for TCR5. Notably, the orientation of DV3 transcription is inverted. So far, it has not been reported that a TCRa chain can utilize a DV3 gene segment.
  • this TCR chain was linked to the identified TCRp chain and then cloned into a retroviral vector. 4171TCR-transduced T cells were strongly reactive to PP-3 ( Figure 9E). This peptide pool PP-3 contained 14 mutated 25- mer-pep tides.
  • LGKFPELFNWFKIFLGYKESVHLET (SEQ ID NO: 25), N>I) was pulsed on autologous DCs for 24 hr. Peptide-pulsed DCs were co-cultured with transduced T cells. The secretion of IFN- ⁇ from T cells was determined by ELISA. 4171TCR-transduced T cells were shown to specifically recognize mutated SIN3 A peptide, but not the wild-type counterpart ( Figure 9G).

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Abstract

L'invention concerne des procédés d'isolement de séquences de chaînes alpha et bêta de récepteur de lymphocytes T (TCR) appariés, ou d'une partie de liaison à l'antigène de ces dernières. L'invention concerne également des procédés d'identification automatique des séquences de segments de chaîne V alpha et bêta de TCR et des séquences CDR3 d'un TCR présentant une spécificité antigénique pour une séquence d'acides aminés mutée codée par une mutation spécifique au cancer. L'invention concerne également des procédés de préparation d'une population de cellules qui expriment des séquences de chaîne alpha et bêta de TCR appariées, ou une partie de liaison à l'antigène de ces dernières. L'invention concerne également des paires isolées de séquences de chaîne alpha et bêta de TCR et des populations isolées de cellules préparées selon les procédés.
PCT/US2018/024828 2017-03-31 2018-03-28 Procédés d'isolement de séquences de récepteur de lymphocytes t spécifiques à un néo-antigène WO2018183485A1 (fr)

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EP23176735.1A EP4234576A3 (fr) 2017-03-31 2018-03-28 Procédés d'isolement de séquences de récepteur de lymphocytes t spécifiques à un néo-antigène
JP2019553512A JP7317712B2 (ja) 2017-03-31 2018-03-28 ネオアンチゲン特異的なｔ細胞受容体配列を単離する方法
US16/495,508 US11898207B2 (en) 2017-03-31 2018-03-28 Methods of isolating neoantigen-specific T cell receptor sequences
CA3057375A CA3057375A1 (fr) 2017-03-31 2018-03-28 Procedes d'isolement de sequences de recepteur de lymphocytes t specifiques a un neo-antigene
AU2018244371A AU2018244371B2 (en) 2017-03-31 2018-03-28 Methods of isolating neoantigen-specific T cell receptor sequences
CN201880022673.3A CN110612446B (zh) 2017-03-31 2018-03-28 分离新抗原-特异性t细胞受体序列的方法
EP18721882.1A EP3602053B1 (fr) 2017-03-31 2018-03-28 Procédés d'isolement de séquences de récepteur de lymphocytes t spécifiques à un néo-antigène
ES18721882T ES2957959T3 (es) 2017-03-31 2018-03-28 Métodos de aislamiento de secuencias de receptores de células T específicos de neoantígenos
CN202310701331.7A CN116726047A (zh) 2017-03-31 2018-03-28 分离新抗原-特异性t细胞受体序列的方法
JP2023117842A JP7569416B2 (ja) 2017-03-31 2023-07-19 ネオアンチゲン特異的なｔ細胞受容体配列を単離する方法
US18/397,448 US20240124935A1 (en) 2017-03-31 2023-12-27 Methods of isolating neoantigen-specific t cell receptor sequences
JP2024174698A JP2025020127A (ja) 2017-03-31 2024-10-04 ネオアンチゲン特異的なｔ細胞受容体配列を単離する方法

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WO2021108727A1 (fr) * 2019-11-27 2021-06-03 Myst Therapeutics, Inc. Procédé de production d'une composition de lymphocytes t réactifs à une tumeur faisant appel à des agents modulateurs
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WO2019084538A1 (fr) * 2017-10-27 2019-05-02 Board Of Regents, The University Of Texas System Anticorps spécifiques à une tumeur, récepteurs de lymphocytes t et procédés d'identification de ceux-ci
US11866785B2 (en) 2017-10-27 2024-01-09 Board Of Regents, The University Of Texas System Tumor specific antibodies and T-cell receptors and methods of identifying the same
WO2020172202A1 (fr) * 2019-02-19 2020-08-27 Myst Therapeutics, Inc. Procédés de production de lymphocytes t autologues utilisés pour traiter les cancers, et compositions associées
EP4394032A3 (fr) * 2019-02-19 2024-10-02 Turnstone Biologics Corp. Procédés de production de lymphocytes t autologues utilisés pour traiter les cancers, et compositions associées
WO2020180648A1 (fr) * 2019-03-01 2020-09-10 Gritstone Oncology, Inc. Sélection de récepteurs de lymphocytes t
WO2020205662A1 (fr) * 2019-03-29 2020-10-08 Myst Therapeutics, Inc. Procédés ex vivo de production d'un agent thérapeutique de lymphocytes t et compositions et procédés associés
WO2021108727A1 (fr) * 2019-11-27 2021-06-03 Myst Therapeutics, Inc. Procédé de production d'une composition de lymphocytes t réactifs à une tumeur faisant appel à des agents modulateurs
WO2025064761A1 (fr) * 2023-09-22 2025-03-27 Regeneron Pharmaceuticals, Inc. Peptides hors cible kras10-18 g12d et leurs utilisations

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